The integrins are a group of adhesion receptors, which play an essential role in cell-cell-binding and cell-extracellular matrix-binding processes. They possess an αβ-heterodimeric structure, have a wide cellular distribution and display a high degree of evolutionary conservation. The integrins include, for example, the fibrinogen receptor on blood platelets, which receptor interacts, in particular, with the RGD sequence of fibrinogen, and the vitronectin receptor on osteoclasts, which receptor interacts, in particular, with the RGD sequence of vitronectin or osteopontin. The integrins are divided into three major groups, i.e., the β2 subfamily, containing the representatives LFA-1, Mac-1 and p150/95, which are responsible, in particular, for cell-cell interactions in the immune system, and the subfamilies β1 and β3, whose representatives principally mediate the adhesion of cells to components of the extracellular matrix (Ruoslahti, Annu. Rev. Biochem., 1988, 57:375). The integrins belonging to the β1 subfamily, which also are called VLA (very late (activation) antigen) proteins, include at least six receptors which interact specifically with fibronectin, collagen and/or laminin as ligands. Within the VLA family, the integrin VLA-4 (α4β1) is a typical, insofar as it is mainly restricted to lymphoid and myeloid cells, and in these cells is responsible for cell-cell interactions with a large number of other cells. For example, VLA-4 mediates the interaction of T lymphocytes and B lymphocytes with the heparin II-binding fragment of human plasma fibronectin (FN). The binding of VLA-4 to the heparin II-binding fragment of plasma fibronectin is based, in particular, on an interaction with an LDVP sequence. In contrast to the fibrinogen receptor or the vitronectin receptor, VLA-4 is not a typical RGD-binding integrin (Kilger and Holzmann, J. Mol. Meth., 1995, 73:347).
Normally, the leukocytes, which are circulating in the blood, only exhibit a low degree of affinity for the vascular endothelial cells, which line the blood vessels. Cytokines, which are released from inflamed tissue, activate endothelial cells and thus the expression of a large number of cell surface antigens. These antigens include, for example, the adhesion molecules ELAM-1 (endothelial cell adhesion molecule 1; also called E selectin), which binds neutrophils, inter alia, ICAM-1 (intercellular adhesion molecule 1), which interacts with LFA-1 (leukocyte function-associated antigen 1) on leukocytes, and VCAM-1 (vascular cell adhesion molecule 1), which binds various leukocytes, inter alia lymphocytes (Osborn et al., Cell, 1989, 59:1203). Like ICAM-1, VCAM-1 is a member of the immunoglobulin gene superfamily. VCAM-1 (first known as INCAM-110) was identified as an adhesion molecule, which is induced on endothelial cells by inflammatory cytokines, such as TNF and IL-1 and lipopolysaccharides (LPS). Elices et al. (Cell, 1990, 60:577) demonstrated that VLA-4 and VCAM-1 form a receptor-ligand pair which mediates the adhesion of lymphocytes to activated endothelium. The binding of VCAM-1 to VLA-4 does not take place here by means of an interaction of the VLA-4 with an RGD sequence since VCAM-1 does not contain such a sequence (Bergelson et al., Current Biology, 1995, 5:615). However, VLA-4 also appears on other leukocytes, and the adherence of leukocytes other than lymphocytes also is mediated by way of the VCAM-1/VLA-4 adhesion mechanism. VLA-4 thus represents a solitary example of a β1 integrin receptor which, by way of the ligands VCAM-1 and fibronectin, plays an essential role both in cell-cell interactions and in cell-extracellular matrix interactions.
The cytokine-induced adhesion molecules play an important role in recruiting leukocytes into extravascular tissue regions. Leukocytes are recruited into inflammatory tissue regions by cell adhesion molecules which are expressed on the surface of endothelial cells and serve as ligands for leukocyte-cell surface proteins or protein complexes (receptors) (the terms ligand and receptor also can be used vice versa). Leukocytes from the blood have first of all to adhere to endothelial cells before they are able to migrate into the synovium. Since VCAM-1 binds to cells, which carry the integrin VLA-4 (α4β1), such as eosinophils, T lymphocytes, B lymphocytes, monocytes and neutrophils, it, and the VCAM-1/VLA-4-mechanism, are responsible for the function of recruiting such cells from the blood stream into infected regions and inflammation foci (Elices et al., Cell, 1990, 60:577; Osborn, Cell, 1990, 62:3; Issekutz et al., J. Exp. Med., 1996, 183:2175).
The VCAM-1/VLA-4 adhesion mechanism has been connected to a number of physiological and pathological processes. In addition to cytokine-induced endothelium, VCAM-1 also is expressed, inter alia, by the following cells: myoblasts, lymphoid dendritic cells and tissue macrophages, rheumatoid synovium, cytokine-stimulated neural cells, parietal epithelial cells of the Bowman's capsule, the renal tubular epithelium, inflamed tissue in connection with heart and kidney transplant rejection, and intestinal tissue in connection with graft-versus-host disease. VCAM-1 also is found to be expressed on those areas of the arterial endothelial tissue, which correspond to early atherosclerotic plaques in a rabbit model. In addition, VCAM-1 is expressed on the follicular dendritic cells in human lymph nodes and is present on stroma cells of the bone marrow, for example, in the mouse. The latter finding suggests that VCAM-1 has a function in B cell development. Apart from cells of hematopoietic origin, VLA-4 also is found, for example, on melanoma cell lines, and the VCAM-1/VLA-4 adhesion mechanism is connected to the metastasis of such tumors (Rice et al., Science, 1989, 246:1303).
The principle form in which VCAM-1 occurs in vivo on endothelial cells, and which is the dominant form in vivo, is designated as VCAM-7D and carries seven immunoglobulin domains. The amino acid sequences of domains 4, 5 and 6 resemble those of domains 1, 2 and 3. The fourth domain is removed, by alternative splicing, in another form, which is composed of six domains and which is designated here as VCAM-6D. VCAM-6D also is able to bind VLA-4-expressing cells.
Further information with regard to VLA-4, VCAM-1, integrins and adhesion proteins can be found, for example, in the articles by Kilger and Holzmann, J. Mol. Meth., 1995, 73:347; Elices, Cell Adhesion in Human Disease, Wiley, Chichester 1995, p. 79 and Kuijpers, Springer Semin. Immunopathol., 1995, 16:379.
On account of the role of the VCAM-1/VLA-4 mechanism in cell adhesion processes, which are of importance, for example, in infections, inflammations and atherosclerosis, attempts have been made to control these diseases, in particular, for example, inflammations (Osborn et al., Cell, 1989, 59:1203), by intervening in these adhesion processes. A method for doing this is the use of monoclonal antibodies, which are directed against the VLA-4. Monoclonal antibodies (Mabs) of this type, which, as VLA-4 antagonists, block the interaction between VCAM-1 and VLA-4, are known. Thus, the anti-VLA-4 Mabs HP2/1 and HP1/3, for example, inhibit the adhesion of VLA-4-expressing Ramos cells (B cell-like cells) to human umbilical cord endothelial cells and to VCAM-1-transfected COS cells. In the same way, the anti-VCAM-1 Mab 4B9 inhibits the adhesion of Ramos cells, Jurkat cells (T cell-like cells) and HL60 cells (granulocyte-like cells) to COS cells, which have been transfected with genetic constructs that cause VCAM-6D and VCAM-7D to be expressed. In vitro data, obtained using antibodies, which are directed against the α4 subunit of VLA-4, show that the adhesion of lymphocytes to synovial endothelial cells, which adhesion plays a role in rheumatoid arthritis, is blocked (van Dinther-Janssen et al., J. Immunol., 1991, 147:4207).
In vivo experiments have demonstrated that anti-α4 Mab can inhibit an experimental autoimmune encephalomyelitis. A monoclonal antibody directed against the α4 chain of VLA-4 likewise blocks the migration of leukocytes into an inflammation focus. The ability of antibodies to exert an effect on the VLA-4-dependent adhesion mechanism also has been examined in an asthma model, in order to investigate the role of VLA-4 in recruiting leukocytes into inflamed lung tissue (WO-A-93/13798). The administration of anti-VLA-4 antibodies inhibited the late phase reaction and the airway hyperreaction in allergic sheep. The significance of VLA-4 as a target for treating asthma is discussed in detail in Metzger, Springer Semin. Immunopathol., 1995,16:467.
The VLA-4 dependent cell adhesion mechanism also has been investigated in a primate model of inflammatory bowel disease (IBD). In this model, which corresponds to ulcerative colitis in humans, the administration of anti-α4 antibodies resulted in a significant reduction in the acute inflammation.
In addition to the conditions discussed above, it has been demonstrated that VLA-4-dependent cell adhesion plays a role in the following clinical conditions, including the following chronic inflammatory processes: rheumatoid arthritis (Cronstein and Weismann, Arthritis Rheum., 1993, 36:147; Elices et al., J. Clin. Invest., 1994, 93:405), diabetes mellitus (Yang et al., Proc. Natl. Acad. Sci. USA, 1993, 90:10494), systemic lupus erythematosus (Takeuchi et al., J. Clin. Invest., 1993, 92:3008), delayed-type allergies (type IV allergy) (Elices et al., Clin. Exp. Rheumatol., 1993, 11:S77), multiple sclerosis (Yednock et al., Nature, 1992, 356:63), malaria (Ockenhouse et al., J. Exp. Med., 1992, 176:1183), atherosclerosis (O'Brien et al., J. Clin. Invest., 1993, 92:945; Shih et al., Circ. Res., 1999, 84:345), transplantation (Isobe et al., Transplantation Proceedings, 1994, 26:867), various malignancies, for example, melanoma (Renkonen et al., Am. J. Pathol., 1992, 140:763), lymphoma (Freedman et al., Blood, 1992, 79:206) and others (Albelda et al., J. Cell Biol., 1991, 114:1059).
The interaction of VLA-4 with VCAM-1 and fibronectin has been connected to some pathophysiological processes in cardiovascular diseases. In an in vitro cell system, immigrated neutrophils inhibit the shortening (negative inotropy) of cardiomyocytes by 35%. It was possible to inhibit this negative inotropic effect of neutrophils by an anti-α4 antibody, but not an anti-CD18 antibody, (Poon et al., Circ. Res., 1999, 84:1245). The importance of VLA-4 in the pathogenesis of atherosclerosis has been demonstrated in a mouse model of atherosclerosis. Thus, the CS-1 peptide, which is directed against the VLA-4-binding site on fibronectin, inhibits the recruiting of leukocytes and the accumulation of fat in the aorta and consequently the formation of atherosclerotic plaques in atherogenically fed LDL receptor-knockout mice (Shih et al., Circ. Res., 1999, 84:345). Using the same CS-1 peptide, it was furthermore possible to show in a heterotopic rabbit heart transplantation model that the formation of a transplant vasculopathy can be significantly reduced by blockade of the interaction of VLA-4 and fibronectin (Molossi et al., J. Clin. Invest., 1995, 95:2601). WO-A-00/02903 describes CS-1 peptidomimetics which contain an aspartic acid unit, or a derivative thereof, in the molecule and which inhibit the binding of VLA-4 to the CS-1 sequence of the matrix protein fibronectin.
Accordingly, blocking VLA-4 by suitable antagonists offers possibilities of achieving an effective treatment, in particular, for example, of treating various inflammatory conditions, including asthma and IBD. The particular relevance of VLA-4 antagonists for treating rheumatoid arthritis follows from the fact that leukocytes from the blood initially adhere to endothelial cells before they are able to migrate into the synovium, and that the VLA-4 receptor plays a role in this adhesion. As mentioned above, inflammatory agents induce VCAM-1 on endothelial cells (Osborn, Cell, 1990, 62:3; Stoolman, Cell, 1989, 56:907), and various leukocytes are recruited into areas of infection and foci of inflammation. In this connection, T cells adhere to an activated endothelium, mainly via the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion mechanisms (Springer, Cell, 1994, 76:301). In rheumatoid arthritis, the binding capacity of VLA-4 for VCAM-1 is increased on most synovial T cells (Postigo et al., J. Clin. Invest, 1992, 89:1445). In addition, an increased adhesion of synovial T cells to fibronectin has been observed (Laffon et al., J. Clin. Invest., 1991, 88:546; Morales-Ducret et al., J. Immunol., 1992, 149:1424). Thus, VLA-4 is up-regulated with respect to its expression and its function on T lymphocytes of the rheumatoid synovial membrane. By blocking the binding of VLA-4 to its physiological ligands VCAM-1 and fibronectin, particular inflammatory processes can be effectively prevented or alleviated. This also is confirmed by experiments, using the antibody HP2/1, which were carried out on Lewis rats suffering from adjuvant arthritis and in which effective disease prevention was observed (Barbadillo et al., Springer Semin. Immunopathol., 1995, 16:427). Thus, VLA-4 is an important therapeutic target molecule.
The abovementioned VLA-4 antibodies, and the use of antibodies as VLA-4 antagonists, are described in the patent applications WO-A-93/13798, WO-A-93/15764, WO-A-94/16094, WO-A-94/17828 and WO-A-95/19790. Patent applications WO-A-94/15958, WO-A-95/15973, WO-A-96/00581, WO-A-96/06108 and WO-A-96/20216 describe peptide compounds, which are VLA-4 antagonists. However, the use of antibodies and peptide compounds as pharmaceuticals suffers from disadvantages, for example, lack of oral availability, easy degradability or an immunogenic action on longer-term administration, and thus there is a need for VLA-4 antagonists possessing a favorable property profile for use in the therapy and prophylaxis of various disease states.
WO-A-95/14008, WO-A-93/18057, U.S. Pat. No. 5,658,935, U.S. Pat. No. 5,686,421, U.S. Pat. No. 5,389,614, U.S. Pat. No. 5,397,796, U.S. Pat. No. 5,424,293 and U.S. Pat. No. 5,554,594 describe substituted 5-membered ring heterocycles, which possess an amino, amidino or guanidino function at the N-terminal end of the molecule and which exhibit platelet aggregation-inhibiting effects. EP-A-796 855 describes other heterocycles, which are inhibitors of bone resorption. EP-A-842 943, EP-A-842 945 and EP-A-842 944 describe that compounds from these series, and other compounds, surprisingly also inhibit leukocyte adhesion and are VLA-4 antagonists.
EP-A-903 353, EP-A-905 139, EP-A-918 059, WO-99/23063, WO-A-99/24398, WO-A-99/54321, WO-A-99160015 and WO-A-00/69831 describe other compounds that inhibit leukocyte adhesion and are VLA-4 antagonists. Further investigations also have shown that the compounds of the present invention surprisingly are strong inhibitors of leukocyte adhesion and antagonists of VLA-4.